Effects of serum and serum-derived factors on growth and differentiation of mouse keratinocytes

Summary Mouse epidermal keratinocytes (MK cells) were grown as replicating subcultures at clonal density, in a serum-free, low calcium basal medium supplemented with seven different growth factors (Bertolero et al., Exp. Cell. Res. 155:64–80, 1984). This serum-free system was used to investigate the activity of cells. bovine serum (FBS) and of serum-derived factors on the growth and differentiation of MK cells. Unfractionated, whole FBS inhibited growth and induced terminal differentiation of normal MK cells. The growth inhibitory activity was considerably reduced by passing whole FBS over a resin (Chelex) to remove Ca²⁺ and other di- and trivalent cations. It is not known whether this treatment removed other factors. Addition of individual serum components either stimulated or inhibited cell-growth and differentiation. Fetuin, a major α-globulin of FBS, and high density lipoprotein strongly inhibited the colony forming efficiency (CFE) of MK cells, whereas bovine serum albumin increased the CFE 4.5-fold and stimulated the growth rate as well. The addition of impure commercial preparations of platelet-derived growth factor inhibited the CFE and induced the morphological features of squamous terminally-differentiating keratinocytes. As reported in other systems, transforming growth factor beta (TGF-β) inhibited the growth of secondary keratinocytes in a dose-dependent manner. Thus, at least three factors present in FBS inhibited growth whereas others were stimulatory. These observations explain the difficulties in obtaining replicating subcultures of mouse keratinocytes in serum-supplemented media and emphasize the importance of a serum-free system for studies on growth control and carcinogenesis in keratinocytes. Editor’s Statement This report contributes significantly to our knowledge of keratinocyte cell biology in two ways. First, a serum-free medium has been developed that can now be used by many investigators to define growth versus differentiation factors for these cells. This is important since several impure or relatively crude preparations of factors are known to influence these cells. Second, the finding that TGF-Beta is an inhibitor of keratinocyte growth opens new avenues to investigate the biochemical events leading to differentiation. David A. Sirbasku     

Growth of preadipocyte cell lines and cell strains from rodents in serum-free hormone-supplemented medium

Summary Ob17 is a clonal cell line isolated from the epididymal fat pad of C57 BL/6J ob/ob mouse that differentiates into adiposelike cells in serum-supplemented medium. In serum-free medium, this cell line shows increased growth under the addition of insulin, transferrin, fibroblast growth factor (FGF), and a factor present in extract of rat submaxillary gland (SMGE). This medium is referred to as 4F. Epidermal growth factor or nerve growth factor cannot replace SMGE, whereas partially purified platelet extract can substitute for FGF but only partially for SMGE. 4F Medium is able to support the proliferation of cells from other established preadipocyte clonal lines, HGFu and 3T3-F442A, and also of preadipocyte cells isolated from the stromal-vascular fraction of rat and mouse adipose tissues. In each case 4F medium is insufficient to support the differentiation of these cells into adipocytes. Ob17 cells grown and maintained in serum-free hormone-supplemented medium retain the ability to convert to adiposelike cells after serum addition. This serum requirement for differentiation cannot be substituted by the addition of growth hormone or of other putative adipogenic factors, or both. The results are discussed with respect to the requirements for growth and differentiation of the 3T3-L1 and 1246 preadipocyte cell lines previously described. This work was supported by the “Centre National de la Recherche Scientifique” (Grant 1208-Biochimie du Développement and Grant 4162-Endocrinologie), by the “Ministère de la Recherce et de la Technologie” (Grant 81-L-1322), by the “Fondation pour la Recherche Médicale,” by NATO (Grant 1704), and by the “Institut National de la Santé et de la Recherche Médicale” (Grant 827006).     

Phenotype alteration in colon carcinoma cells: Effect of in vivo passage?

Summary A panel of rat colon adenocarcinoma cell lines (the Per series) were used to investigate the phenotype and karyotype changes induced by in vivo passage in the subcutis of athymic nude mice. One poorly and one well-differentiated tumor cell line were serially passaged through the athymic nude mouse and then back to the syngeneic rat host. Each of the primary and xenograft cell lines expressed fetal crypt cell (“CaCo”) antigens. The well differentiated primary and xenograft lines (Per305, Per305N1, and Per305N2a) were different in each of their growth factor reponsiveness in vitro [i.e. epidermal growth factor (EGF), bombesin, vasoactive intestinal peptide], their EGF receptor expression, their secretion of transforming growth factor-α, and their exhibition of anchorage independent (A-I) growth capabilities. The poorly differentiated primary and xenograft cell lines were also different but were all capable of A-I growth; their responsiveness to exogenous growth factor stimulation decreased with progressive in vivo passage, as did their basal unstimulated proliferation rate. Cytogenetic alterations detected were those associated with clinical specimens from various stages of malignancy, i. e. aneuploidy, structural aberrations, and marker chromosomes. Genetic and mitogenic individuality of each line demonstrated the diversity of the growth control mechanisms in neoplasms at different stages of progression. Financial support was provided from the Richard Walter Gibbon Fund of the Faculty of Medicine, the University of Western Australia; and from the Sir Charles Gairdner Hospital Research Foundation.     

Evaluation of the role of extracellular matrix proteins,polyunsaturated fatty acids and C-MYC expression in the inhibition of the serum-free growth of epithelial cells by TGF-β1

Summary Novel or modified serum-free media were developed for the anchorage-dependent growth of nontransformed murine mammary epithelial cells (MMEC) and Balb/MK murine keratinocytes respectively. Growth rates for both cell lines were similar in serum-containing and serum-free media. The serum-free media were used to evaluate potential mechanisms of epithelial cell growth regulation by type 1 transforming growth factor β(TGF-β1). The growth of MMEC and Balb/ MK cells was reversibly inhibited 40–65% in a time- and dose-dependent fashion by TGF-β1 under both serum-containing and serum-free conditions. Constitutive over-expression of a stranfected c-myc oncogene inMMEC did not result in loss of sensitivity to growth inhibition by TGF-β1. In addition, Balb/MK and MMEC growth inhibition by TGF-β1 was not potentiated by polynsaturated fatty acids or reversed by vitamin E. Expgenous type V collagen was able to mimic the inhibitory effects of TGF-β1 on the serum-free growth of Balb/MK and MMEC. In contrast, collagen type I and IV, fibronectin and laminin did not inhibit the growth of these cells. The type V collagen used was not contaminated with TGF-β, and subsaturating, but not saturating concentrations of type V collagen and TGF-β1 were additive with respect to Balb/MK and MMEC growth inhibition. These results demonstrate that nontransformed epithelial cell growth inhibition by TGF-β1 is mediated by mechanisms distinct from those observed with certain carcinoma and melanoma cells. Our results also suggest the possible involvement of type V collagen in Balb/MK and MMEC growth inhibition by TGF-β1. This work was supported, in part, by grant #R29 CA 44741 from the National Institutes of Health, Bethesda, MD to NTT.     

The origin and characteristics of a pig kidney cell strain,LLC-PK1

Summary A stable epithelial-like pig kidney cell strain has been established. This strain has been carried through more than 300 serial passages, has remained free of microbial and viral contaminants, and has retained a near diploid number of chromosomes. Attempts to produce tumors with these cells in immunosuppressed laboratory animals have been uniformly negative. The cells have grown rapidly in monolayer cultures with a split ratio of 1 to 15 at weekly intervals, but have failed to proliferate in suspension cultures. A subline adapted to growth on serum-free medium 199 has been carried through 145 passages on this medium. Several unusual morphologic features have been observed in these cultures including three-dimensional “domelike” structures. These cells have been found susceptible to some viruses and have been especially useful for viruses of domestic animals. LLC-PK₁ cells have produced significant levels of plasminogen activator. Histopathological examinations of animal tissues were done by Dr. C. G. Culbertson. Tests for mycoplasma were performed by Mr. D. H. Holmes.     

Immortalization and malignant transformation of Eukaryotic cells

The process of cellular transformation has been amply studied in vitro using immortalized cell lines. Immortalized cells never have the normal diploid karyotype, nevertheless, they cannot grow over one another in cell culture (contact inhibition), do not form colonies in soft agar (anchorage-dependent growth) and do not form tumors when injected into immunodeficient rodents. All these characteristics can be obtained with additional chromosome changes. Multiple genetic rearrangements, including whole chromosome and gene copy number gains and losses, chromosome translocations, gene mutations are necessary for establishing the malignant cell phenotype. Most of the experiments detecting transforming ability of genes overexpressed and/or mutated in tumors (oncogenes) were performed using mouse embryonic fibroblasts (MEFs), NIH3T3 mouse fibroblast cell line, human embryonic kidney 293 cell line (HEK293), and human mammary epithelial cell lines (mainly HMECs and MCF10A). These cell lines have abnormal karyotypes and are prone to progress to malignantly transformed cells. This review is aimed at understanding the mechanisms of cell immortalization by different “immortalizing agents”, oncogene-induced cell transformation of immortalized cells and moderate response of the advanced tumors to anticancer therapy in the light of tumor “oncogene and chromosome addiction”, intra-/intertumor heterogeneity, and chromosome instability.     

High density lipoproteins and the growth of vascular endothelial cells in serum-free medium

Summary Low density bovine vacular endothelial cell cultures maintained on dishes coated with an extracellular matrix can be grown in serum-free Dulbecco's modified Eagle's medium supplemented with high density lipoprotein (HDL) and transferrin. Such cultures do not require insulin. Early passage cultures exposed to HDL and transferrin grew as well as cultures exposed to optimal serum concentrations and could be passaged repeatedly in total absence of serum. A requirement for fibroblast growth factor to ensure an optimal growth could be observed only with late-passage cultures. The present results suggest strongly that HDL is involved in supporting the proliferation of vascular endothelial cells in vitro. This may be important for our understanding of the biological role of HDL “in vivo”. This work was supported by Grants HL 23678 and 20192 from the National Institutes of Health, Bethesda, MD.     

Isolation and Comparative Characteristics of Mesenchymal Stem-Cell Lines Derived from Foreskin of Two Donors of Similar Age

Two new nonimmortalized human cell lines FRSN-1 and FRSN-2 were established from foreskin of two similarly aged donors (2.5 years). Growth characteristics and differentiation potential of these cell lines studied on the sixth passage confirmed their status as mesenchymal stem cells (MSCs). A number of characteristics have been analyzed during long-term cultivation up to the 26th passage. The dynamics of the process of replicative senescence defined by the activity of β-galactosidase differed between these lines. However, at the 26th passage, the process of replicative senescence was equally enhanced in both lines. The plating efficiency markedly differed between the lines on the sixth passage. In FRSN-1, it was higher than in FRSN-2. The plating efficiency substantially dropped to the 26th passage in FRSN-1 and was lost in FRSN-2 line. Growth curves showed active proliferation of these lines at the 6th passage. The average doubling time did not differ between the lines and was 36.9 and 39.0 h, respectively. Analysis of growth curves on the 26th passage revealed a decline in proliferative activity and increase in average doubling time of cell populations in both lines, more in FRSN-2 than in FRSN-1 lines. The patterns of growth curves differed in these lines. Morphological analysis revealed increased cell size and spreading typical for the phase of the replicative senescence. Numerical and structural karyotypic analysis at the sixth passage showed that both lines have normal karyotype 46, XY. We did not discover interline differences in the frequency of chromosomal aberrations. To determine the status of these cell lines, comparative analysis of the surface markers was performed using flow cytometry. It was revealed that cells of both lines expressed surface antigens characteristic of human MSCs: CD44, CD73, CD90, CD105, and HLA-ABC and did not express CD34, CD45 and HLADR. Cells of both lines displayed SSEA-4 and SOX2, markers of human embryonic stem cells (ESCs). Expression of SSEA-4 was also detected at the 26th passage in both lines. FRSN-1 and FRSN-2 cells expressed the markers of early ESC differentiation into three germ layers. The ability of these cell lines to differentiate into osteogenic, chondrogenic, and adipogenic lineages was shown on the sixth passage. Both lines exhibited substantially reduced adipogenic potential on the 20th passage. These data indicate that in contrast to growth characteristics the adipogenic differentiation potential changes even with an average degree of replicative senescence. It appears that the cell replicative senescence contributed to the change in MSC differentiation potential. Overall, the results demonstrate that cell lines derived from different donors are distinguished in growth characteristics and pattern of replicative senescence. The disparity is due to a direct genetic influence and indirectly by different microenvironment in their donor organisms before cell isolation.     

A simple chromosomal marker can reliably distinguishes <Emphasis Type="Italic">Poncirus</Emphasis> from <Emphasis Type="Italic">Citrus</Emphasis> species

Several chromosome types have been recognized in Citrus and related genera by chromomycin A₃ (CMA) banding patterns and fluorescent in situ hybridization (FISH). They can be used to characterize cultivars and species or as markers in hybridization and backcrossing experiments. In the present work, characterization of six cultivars of P. trifoliata (“Barnes”, “Fawcett”, “Flying Dragon”, “Pomeroy”, “Rubidoux”, “USDA”) and one P. trifoliata × C. limonia hybrid was performed by sequential analyses of CMA banding and FISH using 5S and 45S rDNA as probes. All six cultivars showed a similar CMA⁺ banding pattern with the karyotype formula 4B + 8D + 6F. The capital letters indicate chromosomal types: B, a chromosome with one telomeric and one proximal band; D, with only one telomeric band; F, without bands. In situ hybridization labeling was also similar among cultivars. Three chromosome pairs displayed a closely linked set of 5S and 45S rDNA sites, two of them co-located with the proximal band of the B type chromosomes (B/5S-45S) and the third one co-located with the terminal band of a D pair (D/5S-45S). The B/5S-45S chromosome has never been found in any citrus accessions investigated so far. Therefore, this B chromosome can be used as a marker to recognize the intergeneric Poncirus × Citrus hybrids. The intergeneric hybrid analyzed here displayed the karyotype formula 4B + 8D + 6F, with two chromosome types B/5S-45S and two D/5S-45S. The karyotype formula and the presence of two B/5S-45S chromosomes clearly indicate that the plant investigated is a symmetric hybrid. It also demonstrates the suitability of karyotype analyses to differentiate zygotic embryos or somatic cell fusions involving trifoliate orange germplasm. During the submission of this paper, we analyzed 25 other citrus cultivars with the same methodology and we found that the chromosome marker reported here can indeed distinguish Poncirus trifoliata from grapefruits, pummelos, and one variegated access of Citrus, besides the previously reported access of limes, limons, citrons, and sweet-oranges. However, among 14 mandarin cultivars, two of them displayed a single B/5S-45S chromosome, whereas in Citrus hystrix D.C., a far related species belonging to the Papeda subgenus, this chromosome type was found in homozygosis. Since these two mandarin cultivars are probably of hybrid origin, we assume that for almost all commercial cultivars and species of the subgenus Citrus this B type chromosome is a useful genetic marker.     

BALB and kirsten murine sarcoma viruses alter growth and differentiation of EGF-dependent BALB/c mouse epidermal keratinocyte lines

Clonal BALB/c mouse epidermal keratinocyte (BALB/MK) cell lines were established in tissue culture. Despite their aneuploid nature, the lines were nontumorigenic, and retained in vitro properties similar to those of primary diploid keratinocytes. These included the constitutive expression of keratin and terminal differentiation in response to a calcium concentration greater than 1.0 mM in the medium. The cells also demonstrated an absolute requirement for nanomolar concentrations of epidermal growth factor (EGF) for their proliferation. BALB or Kirsten murine sarcoma viruses are acute transforming retroviruses, which have been shown to transform fibroblastic and hematopoietic cells. Infection of BALB/MK or its clonal sublines with either virus leads to the rapid acquisition of EGF-independent growth. The cells concomitantly lose their sensitivity to calcium-induced terminal differentiation. Thus these retroviruses can rapidly confer upon epithelial keratinocytes in culture growth properties that resemble those of malignant epidermoid carcinoma cells.     

Growth modulation of human tumor cells by a growth-inhibiting activity derived from tumorigenic V79 chinese hamster cells

Summary A growth-inhibiting activity was identified in supernatants of the neoplastic V79 Chinese hamster cell line based on its ability to inhibit the proliferation of the same cell line. The partially purified activity, provisionally termed “growth inhibiting factor” (GIF) activity, inhibited the growth of a wide variety of human tumor cells, but not various normal human fibroblasts. This species-nonspecific activity was reversible, saturable, and highly potent in tumorigenic cell lines, and was noted in both monolayer culture and in soft agar. The inhibitory activity CIF was also exhibited in a chemically defined serum-free medium supplemented with insulin and transferrin. GIF activity was stable to acid, heat, trypsin, and dithiothreitol but sensitive to alpha-chymotrypsin. The pattern of growth modulation by GIF on V79 cells was apparently different from those exhibited by bifunctional peptides such as transforming growth factor-beta, tumor necrosis factor-alpha, and interleukin-1-alpha. In addition, GIF activity cannot be ascribed to these cytokines based on the physicochemical and immunologic properties. Although GIF has yet to be purified to homogeneity, these data suggest that GIF might be a novel growth regulator which has a critical role in regulating growth of V79 cells. The growth modulation of tumor cells by this tumor-derived growth inhibiting activity suggested the presence of an autocrine growth regulatory mechanism even in tumor cells.     

Co-culture of primary pulmonary cells to model alveolar injury and translocation of proteins

Summary Primary rat alveolar type II cells and early passage rat lung fibroblasts were co-cultured on opposite sides of a collagen-coated polycarbonate filter. This is an approach to “model”, in part, an alveolar wall to study mechanisms of cytotoxicity and translocation of bioactive materials from the alveolar space to the lung interstitium. Type II cells were recovered from adult rat (Fischer 344) lungs by enzyme digestion and “panning”. Lung fibroblasts were separated from the same species, cultured initially in 10% fetal bovine serum and used in the co-culture system at early passage. The type II cells formed a monolayer of defferentiated epithelium which provided a barrier on the upper side of the collagen (human type IV)-coated filter. The fibroblasts on the bottom of the filter replicated logarithmically in the presence of serum, could be rendered quiescent in defined medium and then returned to rapid growth phase with the reintroduction of serum. The intact epithelial monolayer excluded trypan blue, albumin, platelet-derived growth factor, and alpha₂-macroglobulin from the lower compartment of the culture chamber. Altering the integrity of the monolayer by a variety of means allowed translocation of these materials through the collagen-coated filters. Particularly interesting was the effect of taurine chloramine which caused subtle changes in the alveolar epithelium and allowed subsequent translocation of albumin. In addition, we showed that rat alveolar macrophages remain viable with some spreading on the surface of the epithelial monolayer. This co-culture system will have future application in the study of how reactive oxygen species might affect the epithelial barrier, and whether macrophage-derived growth factors can influence fibroblast proliferation if the monolayer is intact or injured.     

小鼠胚胎干细胞(ES-8501细胞)建系过程的核型及特性分析

小鼠胚胎性癌(EC)细胞系的细胞核型大多数异常,对用于分析EC细胞与胚胎细胞之间的关系和进行嵌合体研究等都是不利的。人们都期望能有正常核型的胚胎细胞系的建立。近年来Evans和Kaufman以及Martin等人先后用不同方法直接从小鼠的内细胞团(ICM)细胞建立了多潜能的胚胎干细胞(erabryonicstem eells,简称ES细胞),也有人称之为EK     

Establishment of the AU-bek cell line and comparison with two other bovine cell lines

Summary Establishment of a new bovine cell line, AU-BEK, is reported. The cell line developed in a culture initiated from bovine embryonic kidneys by spontaneous cultural alteration to epithelioid cells that are indefinitely propagable. Epithelioid cells gradually increased to become the predominant cell. Whereas normal bovine cells have a diploid number of 60 chromosomes, of which only the two sex chromosomes are biarmed, AU-BEK cells at the 80th passage had a modal chromosome number of 84 and an average of 30 biarmed chromosomes per cell. AU-BEK cells are now in their 220th passage. Of the AU-BEK, MDBK, and CKT-1 bovine cell lines, the CKT-1 cell line had a karyotype closest to that of normal bovine cells. Their modal chromosome number was 57, and only three biarmed chromosomes were usually present. The bovine character of AU-BEK and CKT-1 cells was established by cytotoxic and viral susceptibility tests. Supported by the Alabama Agricultural Experiment Station. Publication No. 1115, School of Veterinary Medicine, Auburn University.     

Slow modulation of synaptic transmission by brain-derived neurotrophic factor leads to the central sensitization associated with neuropathic pain

Chronic constriction injury (CCI) of the rat sciatic nerve increases the dorsal horn excitability. This “central sensitization” leads to behavioral manifestations analogous to those related to human neuropathic pain. We found, using whole-cell recording from acutely isolated spinal cord slices, that 7-to 10-day-long CCI increases excitatory synaptic drive to putative excitatory “delay”-firing neurons in the substantia gelatinosa but attenuates that to putative inhibitory “tonic”-firing neurons. A defined-medium organotypic culture (DMOTC) system was used to investigate the long-term actions of brain-derived neurotrophic factor (BDNF) as a possible instigator of these changes. When all five neuronal types found in the substantia gelatinosa were considered, BDNF and CCI produced similar patterns, or “footprints,” of changes across the whole population. This pattern was not seen with another putative “pain mediator,” interleukin 1β. Thus, BDNF decreased synaptic drive to “tonic” neurons and increased synaptic drive to “delay” neurons. Actions of BDNF on “delay” neurons were presynaptic and involved increased mEPSC frequency and amplitude without changes in the function of postsynaptic AMPA receptors. By contrast, BDNF exerted both pre-and post-synaptic actions on “ tonic” cells to reduce the mEPSC frequency and amplitude. These differential actions of BDNF on excitatory and inhibitory neurons contributed to a global increase in the dorsal horn network excitability as assessed by the amplitude of depolarization-induced increases in the intracellular [Ca²⁺]. Experiments with the BDNF-binding protein TrkB-d5 provided additional evidence for BDNF as a harbinger of neuropathic pain. Thus, the cellular processes altered by BDNF likely contribute to “central sensitization” and hence to the onset of neuropathic pain. Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 315–326, July–October, 2007.     

Black rat (Rattus rattus) genomic variability characterized by chromosome painting

Black rats are of outstanding interest in parasitology and infective disease analysis. We used chromosome paints from both the mouse(Mus musculus) and the Norway rat(Rattus norvegicus) to characterize the genome of two Black rat subspecies from Italy. Both subspecies have two large metacentrics (n. 1, 4) not present in the Norway rat (2n = 42).Rattus rattus rattus has a diploid number of 2n = 38, whileRattus rattus frugivorous has two small metacentric “supernumerary” or B chromosomes for a diploid number of 2n = 38 + 2B. The 21 mouse paints gave 38 signals on theR. r. rattus karyotype and 39 signals in theR. r. frugivorous karyotype. The two metacentrics, not present inR. norvegicus, were hybridized by mouse 16/1/17 and mouse 4/10/15. These chromosomes are homologous to: RRA1 = RNO 5/7, and RRA4 = RNO 9/11 and not “4/7” and “11/12” as previously reported. Furthermore, the synteny of Chr 13 of theR. r. frugivorous withR. norvegicus Chr 16 and mouse Chrs 8/14 is not complete, because there is a small pericentromeric insertion of RNO Chr 18 (mouse Chr 18). If we consider only the two metacentrics, RRA1 and RRA4, the principal differences betweenR. norvegicus andR. rattus, then we can propose the derived synteny of 124 genes in the black rat. A comparison of the Z index between rats and mice shows an acceleration of genomic evolution among genus, species, and subspecies. The chromosomal differences betweenR. r. rattus xR. r. frugivorous suggest that they may be classified as different species because hybrids would produce 50% unbalanced gametes.     

Indian rice “Kasalath” contains genes that improve traits of Japanese premium rice “Koshihikari”

Rice (Oryza sativa L.) chromosome segment substitution lines (CSSLs), in which chromosomal segments of the Indian landrace “Kasalath” replace the corresponding endogenous segments in the genome of the Japanese premium rice “Koshihikari”, are available and together cover the entire genome. Chromosome regions affecting a trait (CRATs) can be identified by comparison of phenotypes with genotypes of CSSLs. We detected 99 CRATs for 15 agronomic or morphological traits. “Kasalath” had positively acting alleles in 53 CRATs. Its CRATs increased panicle number per plant by up to 23.3%, grain number per panicle by up to 30.8%, and total grain number by up to 15.1%, relative to “Koshihikari”. CRATs were identified for grain size (grain thickness and width), with positive effects of about 5.0%. A CRAT on chromosome 8 almost doubled the weight of roots in uppermost soil layers compared to “Koshihikari”. Additionally, “Kasalath” possessed CRATs for higher lodging resistance (reduction in plant height and increase in stem diameter). In some cases, multiple CRATs were detected in the same chromosome regions. Therefore, CSSLs with these chromosome segments might be useful breeding materials for the simultaneous improvement of multiple traits. Five CRATs, one for plant height on chromosome 1, one for stem diameter on chromosome 8, and three for heading date on chromosomes 6, 7, and 8 overlapped with the corresponding QTLs that already had been mapped with back-crossed inbred lines of “Nipponbare” and “Kasalath”. In both “Koshihikari” CRATs and “Nipponbare” QTLs, “Kasalath” had similar effects. Both Y. Madoka and T. Kashiwagi have contributed equally to this article.     

Establishment and characterization of an immortalized but non-transformed human prostate epithelial cell line: BPH-1

Summary This report describes the development and characterization of an epithelial cell line (BPH-1) from human prostate tissue obtained by transurethral resection. Primary epithelial cell cultures were immortalized with SV40 large T antigen. One of the isolated clones was designated BPH-1. These cells have a cobblestone appearance in monolayer culture and are non-tumorigenic in nude mice following subcutaneous injection or subrenal capsule grafting. They express the SV40 large T antigen and exhibit increased levels of p53, as determined by immunocytochemistry. Cytogenetic analysis by G-banding demonstrated an aneuploid karyotype with a modal chromosome number of 76 (range 71 to 79,n=28) and 6 to 8 marker chromosomes. Some structurally rearranged chromosomes were observed, but the Y chromosome was normal. The expressed cytokeratin profile was consistent with a prostatic luminal epithelial cell. This profile was the same as that of primary prostatic epithelial cultures from which the BPH-1 cells were derived. In serum-free culture in plastic dishes epidermal growth factor (EGF), transforming growth factor (TGF)-α, fibroblast growth factor (FGF) 1 (aFGF), and FGF 7 (KGF) induced increased proliferation in these cells whereas FGF 2 (bFGF), TGF-β1, and TGF-β2 inhibited proliferative activity. Testosterone had no direct effect on the proliferative rate of BPH-1 cells. 5α-Reductase, 3α-hydroxysteroid oxidoreductase, and 17β-hydroxy-steroid oxidoreductase activities were detected in BPH-1 cells. Expression of androgen receptors and the secretory markers, prostate specific antigen and prostatic acid phosphatase, were not detectable by immunocytochemistry, biochemical assay, or RT-PCR analysis.     

Cytogenetic study of three edible chrysanthemum cultivars

During past decades, edible chrysanthemum (Dendranthema × grandiflorum) has become a popular dietary supplement. However, only numberable cultivars are available, cytogenetic studies especially meiotic behaviour in them remaind largely unexplored. In the present study, we analyzed the karyotype and meiotic behaviour during microsporogenesis in accessions of edible chrysanthemum. This information can be useful in cultivar improvement, as well as in elucidation of the evolution of the species. The three edible cultivars studied all had an intermediate type of interphase nucleus, and their mitotic prophase chromosomes were of the interstitial type. The chromosome number of “Baohuatangyijin”(Bn) varies from 55 to 62. “Zifengmudan”(Zn) has the karyotype 2n = 54 = 38m + 12sm + 4st, while “Jingxingxiying”(Jg) has 2n = 55 = 38m + 15sm + 2st. Male meiosis was largely normal. Mean pairing configuration of Zn at diakinesis and metaphase I was 0.94I + 25.07II + 0.14III + 0.63IV, while for Jg, the equivalent was 1.32I + 24.64II + 0.16III + 0.85IV + 0.05V + 0.04VI. Chromatid separation was normal during anaphase I and anaphase II in most meiocytes. All three edible chrysanthemums appear to be allopolyploid, and the edible type is probably more primitive than the ornamental type. However, the edible type is probably more closely related to the ornamental than to the medicinal type.     

Nuclear-cytoplasmic interactions in restoration of male fertility in the ‘9E’ and A4 CMS-inducing cytoplasms of sorghum

 The genetics of male-fertility restoration in sorghum in the “9E” and A4 CMS-inducing cytoplasms, was studied by crossing a number of fertility restorer lines of A1 cytoplasm to CMS lines [9E]T×398 and [A4]T×398 and the line [9E]Milo-10, which was obtained by backcrossing Milo-10 to [9E]T×398. It was revealed that both A4 and “9E” cytoplasms are characterized by a sporophytic mode of restoration of male fertility. Depending on the nuclear background of the male parents, fertility restoration was controlled by one or two dominant genes. Fertility-restorer genes of one of the tester lines, KVV-114, were effective in [9E]T×398 but could not restore [9E]Milo-10. A fertile line obtained from the fertile hybrid [9E]T×398/KVV-112, with “9E” cytoplasm, also failed to restore [9E]Milo-10. In a number of hybrid combinations with both A4 and “9E” cytoplasms a novel and unusual phenomenon of gradual restoration of male fertility in subsequent backcross generations was observed. Pollen from the fertile revertants did not transmit fertility restoration in progeny from crosses with the original CMS line and was poorly transmitted in sib-crosses. The appearance of fertile revertants and the different reactions of different CMS lines with the same cytoplasm in test-crosses may be caused by the action of recessive nuclear genes of the recurrent male parents that were accumulated during backcrossing; these may induce changes in cytoplasmic genes controlling CMS. Received: 5 March 1998 / Accepted: 7 April 1998